Numerous assays have been developed for the detection and determination of proteins, e.g., antibodies in biological fluids. One class, the immunoassays, which has evolved into an invaluable tool in diagnostics, is based upon the principle of specific binding of antibodies to haptens and/or antigens. In a typical immunoassay, a test sample, containing an analyte of interest, and an antibody, to which it specifically binds, are incubated, and then washed to separate free and bound analytes. An enzyme-labeled antibody that recognizes the resulting complex is added, incubated, and washed, and finally substrate, an enzyme detection system, is added and the labeled complex is detected and determined.
Conjugates of peptides specific to antibodies have been used advantageously in immunoassays. Conjugates of peptide analogues of viral proteins, that is, segments of the proteins bearing the epitopic sequence, for example, of the human immunodeficiency viruses (HIV) and labeled enzymes linked through a maleiimide moiety have been described as being particularly advantageous in the detection and determination of antibodies to HIV. See U.S. Pat. No. 5,294,536 granted to Paul S. Palumbo on Mar. 15, 1994 ('536-patent). The conjugates of activated peptides, prepared by the processes described in the '536- patent are not homogenous, the activated peptides being derived from the terminal amino group and/or the internal amino and hydroxyl groups of the peptide analogue. Interaction of the internal amino and/or hydroxyl groups of the peptide analogue and the crosslinking agent, in addition to that of the terminal amino group, diminishes the effectiveness of the epitopic centers of the peptide analogue thereby reducing the sensitivity of the assay for the detection and determination of specific binding antibodies. For example, interaction of the amino group of the lysine subunit and/or the amino and the hydroxy subunits of the serine subunit of the epotopic segment of the peptide analog of the HIV virus and the crosslinker reduces the sensitivity of the immunoassay for the detection or determination of antibodies to HIV, the greater the interaction of the internal amino and/or hydroxyl group relative to the terminal amino group, the lower the sensitivity of the assay. The processes for the preparation of activated peptides 3 and conjugates 5 described in the aforementioned '536-patent involve condensation of an isocyanatomaleiimide 1 ##STR1##
wherein X is a spacer group with a peptide having a terminal amino group of formula 2 EQU RNH.sub.2 2
wherein R is the remainder of the peptide to form an activated peptide 3 ##STR2##
wherein R and X are as above, which is then condensed with a thiolated enzyme of formula 4 EQU R.sub.1 SH 4
wherein R.sub.1 is the remainder of an enzyme to form a conjugate of formula 5 ##STR3##
Wherein X, R, and R.sub.1 are as defined herein. The critical step in the process, the reaction of the crosslinker 1 with a peptide 2, characterized by the presence of a terminal amino group and internal amino and/or hydroxyl groups, affords an activated peptide 1 derived from the terminal amino group, as well as activated peptides derived from the internal amino and/or hydroxyl groups of the peptide, and combinations thereof.